Fabrication of RGD peptide gradient poly(ethylene glycol) (PEG) hydrogel in microfluidic gradient generators to control mesenchymal stem cell behaviour

In the design of biomaterial interfaces to control cell response, efforts have focused on tuning surface chemistry to control the concentration or gradient of biochemical agents. The microfluidic methods have been used to precisely control the gradient profiles to study the different kinds of cells response. However, few studies have been explored for the Mesenchymal Stem Cell (MSC) response to small biomolecules concentration using the microfluidic gradient methods, especially in a 3D environment. MSCs have the potential use for tissue regeneration in three-dimensional (3D) scaffold through the control and guidance of MSCs differentiation. The purpose of this project is to fabricate the poly (ethylene glycol) (PEG) hydrogel with gradient distribution of Arg-Gly-Asp (RGD) peptide using the microfluidic gradient generator to study Rat MSC adhesion on the surface of 2D PEG hydrogel and cell viability in 3D environment by cell encapsulation.;The microfluidic gradient generator was fabricated using photolithography and soft lithography technique. Colour solution was used to test the formation of gradient in the generator. PEG hydrogel with RGD gradient was then fabricated in the microfluidic device under UV light irradiation. Rat MSC was cultured on the 2D RGD gradient PEG hydrogel. Actin and nucleus staining were done to observe cell adhesion and spreading. MSC was also 3D encapsulated into the RGD gradient PEG hydrogel under UV light. The cells viability was characterized by live/dead assay. The encapsulated cells were induced to osteogenic differentiation.;Theoretical simulation and colour solution testing show that chemical gradient can be successfully generated in the microfluidic device. MSC cultured on 2D RGD gradient PEG hydrogel had a gradient distribution that cell adhesion density and covering area increased with the increase of RGD concentration. The viability for 3D encapsulation also increased with the increase of RGD concentration. Characterization of osteogenic differentiation in the RGD gradient hydrogel was also done and results indicated that RGD can promote the osteogenic differentiation.